Proper cellular function requires efficient folding of cellular proteins. In the cell, protein foldng starts cotranslationally, concomitantly with protein synthesis by the ribosome, following pathways that can be distinct from the refolding of full-length proteins in vitro. The average, bul translation rate in E. coli is -20 aa/sec, but this rate can vary by more than an order of magnitude for the translation of specific mRNA sequences. Recent results have highlighted that altering the translation rate of small portions of some genes can significantly affect the folding efficiency of the encoded protein (correct folding versus misfolding and aggregation, or degradation), or alter the partitioning between two alternative folded structures. Yet despite the potential impact of local translation rate on protein biogenesis in vivo, the interactions between the ribosome and mRNA and/or nascent chain sequences that control local translation rate remain opaque. Nor do we know what fraction of proteins in the proteome has co-translational folding mechanisms that are significantly affected by altered translation rate. The Pis of this proposal have constructed a network to span their established expertise in genetics, molecular biology, genomics, biochemistry, biophysics, and organic, analytical and physical chemistry, creating a team uniquely suited to tackle three significant outstanding questions regarding the mechanisms and outcomes of altered translation rate in E. coli. Our network will determine: (1) What specific features of mRNA and/or nascent chain sequences shape absolute local translation rate in vivo, and by what mechanisms? (2) What proteins in the proteome are most susceptible to aggregation or degradation when translation rate is altered? (3) To what extent does translational pausing alter the conformation of the ribosome and/or its interactions with other proteins in vivo? Taken together, results from this proposal will represent crucial steps towards a comprehensive picture of translation rate control in E. coli, and the effects of altered translation rate on protein biogenesis, including the heterologous expression of human proteins. PUBLIC HEALTH RELEVENCE: The rate of protein synthesis is not uniform, but we do not fully understand why some sequences are translated more slowly than others. Altering local translation rate can affect protein folding, and hence cell function. We will develop new approaches to measure local translation rates and the mechanisms that determine these rates, and identify proteins whose folding is most affected by translation rate differences.